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. 2015 Apr;4(4):e26.
doi: 10.1038/emi.2015.26. Epub 2015 Apr 22.

A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus

Yixin Chen  1 , Kwok-Hung Chan  2 , Yahong Kang  1 , Honglin Chen  3 , Hayes K H Luk  2 , Rosana W S Poon  2 , Jasper F W Chan  3 , Kwok-Yung Yuen  3 , Ningshao Xia  1 , Susanna K P Lau  3 , Patrick C Y Woo  3
Affiliations

A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus

Yixin Chen et al. Emerg Microbes Infect. 2015 Apr.

Abstract

Since its emergence in 2012, more than 900 laboratory-confirmed cases of Middle East respiratory syndrome (MERS) have been reported with a fatality rate of more than 30%. However, no antigen detection assay for commercial use is available for diagnosis. In this study, the full-length nucleocapsid protein (NP) gene of MERS coronavirus (MERS-CoV) was cloned and expressed in Escherichia coli. A MERS-CoV NP capture enzyme-linked immunosorbent assay (ELISA) using two MERS-CoV-NP-specific monoclonal antibodies (MAbs) generated was developed. The ELISA was evaluated using 129 nasopharyngeal aspirates (NPAs) positive for various respiratory viruses and simulated positive NPAs by adding serial dilutions of MERS-CoV. Using a cutoff OD of 0.19, all 129 NPAs positive for respiratory viruses showed very low OD, with a specificity of 100%. For the two simulated MERS-CoV-positive NPAs with serial dilutions of live MERS-CoV, all samples with ≥10 50% tissue culture infective dose (TCID50)/0.1 mL showed positive results. For the 10 additional NPAs with 20 and 200 TCID50/0.1 mL of live MERS-CoV added, all were positive. A highly sensitive and specific MAbs-based antigen capture ELISA has been developed for MERS. This sensitive and specific antigen capture ELISA should be useful for detection of MERS-CoV in human and dromedaries and in field studies.

Keywords: ELISA; MERS coronavirus; antigen; detection.

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Figures

Figure 1
Figure 1
Western blot analysis of mouse anti-MERS-CoV-rNP immune sera. Proteins loaded include: MERS-CoV-rNP, lysate of MERS-CoV (MERS-CoV), lysate of influenza A virus strain CA/04 (Flu CA/04), molecular weight marker. The arrows indicate the size of the MERS-CoV-rNP.
Figure 2
Figure 2
The ELISA titer of mouse anti-MERS-CoV-rNP immune sera. The MERS-CoV-rNP was coated at 100 ng/well. 1:5000 dilution of HRP conjugated Goat anti-mouse antibody was used as second antibody. Normal mouse sera were used as negative control.
Figure 3
Figure 3
Reactivity of anti-MERS-CoV-rNP MAbs in ELISA. The MERS-CoV-rNP was coated at 100 ng/well. 1:5000 dilution of HRP-conjugated goat anti-mouse antibody was used as the secondary antibody. Normal mouse sera were used as negative control.
Figure 4
Figure 4
Detection limit of anti-MERS-CoV-rNP-specific MAbs-based capture ELISA for MERS-CoV-NP. (A) Detection of MERS-CoV-rNP, HCoV-OC43-rNP, and HCoV-229E-rNP. (B) Detection of inactivated cell lysates of MERS-CoV, HCoV-OC43, HCoV-229E, influenza A virus CA/4/2009(H1N1), influenza A virus Brisbane/10/2007(H3N2), influenza B virus Florida/04/2006, and RSV cultures.
Figure 5
Figure 5
Evaluation of the MERS-CoV-NP capture ELISA using 129 NPAs positive for various viral respiratory tract infections, two simulated MERS-CoV-positive NPAs spiked with serially diluted MERS-CoV cell lysates and 10 simulated MERS-CoV-positive NPAs with 20 TCID50/0.1 mL and 200 TCID50/0.1 mL of MERS-CoV. The dotted line represents the corresponding cutoff OD450 value at 0.19.
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